Tumor markers are molecules that are associated with the transformation of a normal cell into a malignant cell. Tumor markers are either altered proteins which are different from the proteins expressed in normal cells or over-expression of proteins that are not expressed, or slightly expressed in normal cells.
Mucins are high molecular weight glycoproteins, which are produced by normal epithelial cells. MUC1 is one of the four mucins known to date that are transmembrane molecules and while its function in adult life maybe lubrication, in fetal life development it is thought to play an important role in forming the lumen of the duct by keeping apart cells located opposite one another. The MUC1 gene was also shown to be expressed in hemopoietic tissues. It was found that MUC1 (also called H23-Ag, episialin, PEM—Polymorphic Epithelial Mucin, MCA—Mammary Carcinoma Antigen and EMA Epithelial Membrane Antigen) expression is elevated (10-50 fold) in breast cancer cells in comparison to normal resting mammary secretory epithelial cells. Moreover, immunohistochemical analyses using the H23 mAb (which recognizes MUC1), revealed that MUC1 is expressed in 91% of the breast cancer tissues and 100% of breast cancer metastases, whereas the non-malignant tissues were negative for the H23 mAb staining (1). Elevated levels of the MUC1 protein in serum and body fluids were reported in 7%, 17%, 64% and 67% of breast cancer patients presenting with stages I to IV, respectively. In addition, elevated levels of circulating MUC1 may be associated with a poor prognosis.
It was shown that MUC1 is over expressed in epithelial cancers other than breast cancer. MUC1 was shown to be over expressed in epithelial ovarian cancer cells as well as in all types of lung cancer cells and other cancers (2).
In malignancy, the MUC1 oligosacharide chains are shorter and less dense comparing to MUC1 in normal cells. This results in the exposure of new epitopes of the core protein in the cancer-associated mucin.
Isolation of MUC1 cDNAs revealed several protein isoforms: the MUC1/REP, MUC1/SEC MUC1/Y and MUC1/X proteins. A short description of four of the above-mentioned isoforms that are connected with the present invention is given below:
The MUC1/REP isoform is a transmembrane protein that contains: a large extracellular domain consisting of a heavily glycosylated 20 amino acid repeat motif. The number of these repeats varies from 20 to 100 and thus is named a VNTR (Variable Number of Tandem Repeats); a transmembrane domain, which consists of a 28 amino acid hydrophobic sequence, and a 72 amino acid cytoplasmic domain. During the biosynthesis of the MUC1/REP protein it undergoes a proteolytic cleavage event. The cleavage takes place within the conserved sequence IKFRPGSWV (SEQ ID NO:3) that is contained within the extracellular domain. Intriguingly, this cleavage site resides within a previously identified module, designated the “SEA” module (3), found in a number of highly 0-linked glycosylated proteins that are invariably linked in one way or another to the cell membrane. The SEA module functions not only as a site for proteolytic cleavage, but also for subsequent re-association of the subunits. Consequently, the MUC1/REP protein is presented on the cell surface as a heterodimer which is composed of a large extracellular subunit (containing the repeat array) linked by non-covalent, SDS sensitive bonds to a smaller cell-anchored subunit which consists of a small extracellular fragment followed by the transmembrane and cytoplasmic domains. The large extracellular subunit can disconnect and reconnect with the small extracellular fragment of the cell-anchored subunit.
The MUC1/Y isoform is a transmembrane protein that contains transmembrane and cytoplasmic domains identical to those of MUC1/REP protein. Unlike MUC1/REP, due to a differential splicing event that utilizes splicing sites located upstream and downstream to the repeat array, MUC1/Y protein is devoid of both the tandem repeat array and its flanking regions. Expression of MUC1/Y was demonstrated in various human secretory epithelial tumors. MUC1/Y was found to be expressed on the cell surface of various human epithelial tumor cells but is not detectable in the adjacent normal tissue.
The MUC1/X isoform (4) is a transmembrane protein that contains transmembrane and cytoplasmic domains identical to those of MUC1/REP protein. Unlike MUC1/REP, due to a differential splicing event that utilizes splicing sites located upstream and downstream to the repeat array, MUC1/X protein is devoid of both the tandem repeat array and its flanking regions.
The MUC1/SEC isoform: This isoform is generated by an alternative splicing mechanism. It is a secreted protein since it lacks the hydrophobic region that can attach the protein to the cell membrane. Its N-terminal sequences are identical to those of the MUC1/REP extracellular domain. Furthermore it has been shown that the soluble secreted MUC1/SEC protein may bind specifically to the extracellular domain of the MUC1/Y protein (5).
Thus, it will be highly advantageous to develop a ligand which binds to a specific epitope in the MUC1 proteins and, more particularly, to a specific extracellular epitope in the MUC1/REP or MUC/Y proteins that will dramatically inhibit the proliferation or growth of cells, and will induce death in cells, such as, cancer cells and in particular cells which over express MUC1 proteins.